The present invention relates generally to a polypeptide of Neisseria meningitidis of approximately 40-55 kD referred to as xe2x80x9cNMASPxe2x80x9d. The invention encompasses an isolated or purified NMASP polypeptide and polypeptides, including fragments, derived therefrom (NMASP-derived polypeptides), and methods of making thereof. The invention also encompasses antibodies, including cytotoxic or bactericidal antibodies, that specifically bind the NMASP polypeptide, NMASP-derived polypeptides and/or fragments thereof. The invention further encompasses immunogenic, prophylactic or therapeutic compositions, including vaccines, that comprise NMASP polypeptide, NMASP-derived polypeptides and/or fragments thereof. The invention additionally provides methods of inducing an immune response to Neisseria meningitidis in an animal and methods of treating infections in an animal caused by Neisseria meningitidis The invention further provides isolated nucleotide sequences encoding the NMASP polypeptide, NMASP-derived polypeptides and fragments thereof, vectors having said sequences, and host cells containing said vectors.
Neisseriae are gram-negative diplococci and include but are not limited to Neisseria ovis, Neisseria lacunata, Neisseria osloensis, Neisseria bovis, Neisseria meningitidis, and Neisseria gonorrhoeae. Neisseria meningitidis (xe2x80x9cN.m.xe2x80x9d) is the most common cause of bacterial meningitidis and septicemia in infants and young adults in the industrialized world; markedly so in countries that have initiated immunization programs against Haemophilus influenzae type B (Hib) disease (Riedo, F. X. et al. 1995. Epidemiology and prevention of meningococcal disease. Pediatr. Infect. Did. J. 14:643-657; Hart, C. A. And T. R. Rogers. 1993. Meningococcal disease. J. Med. Microbiol. 39:3-25; Jackson, L. A. And J. D. Wenger. 1993. Laboratory-based surveillance for meningococcal disease in selected areas, United States, 1989-1991. MMWR 42:21-30). World-wide, N. meningitidis accounts for about ⅓ of all cases of bacterial meningitis; with most countries showing an attack rate of  greater than 1/100,000 population. Mortality as a whole is significantly higher with the meningococci than with Hib disease. Unlike Hib infections which are basically sporadic limited outbreaks, epidemics of meningococcal disease occur regularly throughout the world and cause great suffering and death. Attack rates during epidemics can exceed 600/100,000 (Hart, C. A. And T. R. Rogers. 1993. Meningococcal disease. J. Med. Microbiol. 39:3-25; Jones, D. 1995. Epidemiology of meningococcal disease in Europe and the USA. In: Meningococcal Disease. Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). Despite the organism""s sensitivity to a wide variety of antibiotics and the impact antibiotic intervention has had on the overall case fatality rate, meningococcal disease attack rates have changed very little since the introduction of antibacterials and the fatality rate still remains between 7 and 15% even in industrialized countries.
N.m. infection starts with colonization of the upper respiratory tract; primarily the tonsils and nasopharynx (Brandtzaeg, P. 1995. Pathogenesis of meningococcal infections. In: Meningococcal Disease. Cartwright, K. (Ed.), Wiley Press, New York, USA: 145-157). Once colonization is established, the organism can invade the underlying endothelium and gain entry into the circulatory system where it causes a rapid, fulminate meningococcemia and/or progresses to the cerebrospinal fluid to cause an often fatal meningitis. To reach the meninges, the organism must interact and circumvent two cellular barriers, the nasopharynx and the blood-brain barrier. Bacterial-host cell interactions are thus critical for the pathogenesis of N.m. Pili, cell surface attachment components, and the polysaccharide capsule all play essential roles in the initial attachment and colonization processes (Jerse, A. E. And R. F. Rest. 1997. Adhesion and invasion by the pathogenic neisseria. Trends Microbiol.:217-221). Once colonization of the upper respiratory tract has been achieved, the organism can down-regulate pili expression and capsule synthesis and expresses other afimbrial adhesins and invasion proteins possibly masked by the capsule that allow the bacteria to invade the underlying endothelial cells.
Based on the structural carbohydrate composition of the meningococcal capsular polysaccharide (CPS), N.m. strains can be divided into a least 12 serogroups, designated types A through L (Riedo, F. X. et al. 1995. Epidemiology and prevention of meningococcal disease. Pediatr. Infect. Did. J. 14:643-657; Hart, C. A. and T. R. Rogers. 1993. Meningococcal disease. J. Med. Microbiol. 39:3-25). However, serogroups A, B, and C account for over 90% of meningococcal disease and are the serotypes most often associated with epidemic disease (Jones, D. 1995. Epidemiology of meningococcal disease in Europe and the USA. In: Meningococcal Disease. Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). In the United States and most developed countries, roughly half of the meningococcal meningitis cases are caused by serogroup B. The highest attack rates of type B meningococcal disease are observed in young children under the age of two with the peak incidence seen in children less than 1 year of age.
The CPS has been targeted as a prime vaccine candidate for the meningococci. Several laboratories have shown that anti-CPS antibodies promote complement-mediated killing of organisms which belong to the same but not different capsular serogroups (Gotschlich, E. C. et al. 1977. The immune responses to bacterial polysaccharides in man. In: Antibodies in Human Diagnosis and Therapy. Haber, E. And R. M. Krause (Eds.), Raven Press, New York, USA: 391-402). The emergence of sulfonamide-resistant organisms in military recruits spurred the development of CPS vaccines against serogroups A, C, and W. While these vaccines are highly immunogenic and effective in adults, the immune response elicited in infants is minimal and of short duration, due primarily to the fact that the very young respond poorly to T-cell-independent antigens like the CPS immunogen.
Prototype serogroup B polysaccharide vaccines have been produced but were found to be poorly immunogenic in humans and gave rise to only low avidity antibody that does not stimulate high levels of complement-mediated killing or opsonization (Frasch, C. E. 1995. Meningococcal vaccines: past, present and future. In: Meningococcal Disease. Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). The poor immunogenicity of the type B CPS is believed to result from the structural similarity of the type B capsule polysaccharide to the sialic acid structures (xe2x80x94xe2x88x922,8 linkage) found on the surface of human brain neural cell glycoproteins (NCAMS) (Finne, J. et al. 1983. Occurrence of alpha-2,8 linked polysialosyl units in neural cell adhesion molecules. Biochem. Biophys. Res. Comm. 112:482-487). The poor immune responsiveness of type B CPS and the possibility that anti-type B capsular antibody may recognize native human carbohydrate structures and possibly trigger an autoimmune sequelae has resulted in a greater emphasis on the evaluation of alternative meningococcal surface antigens as potential vaccine candidates (Poolman, J. T., et al. 1986. Class ⅓ outer membrane protein vaccine against group B, type 15, subtype 16 meningococci. Dev. Biol. Stand. 63:147-152; Ala""Aldeen, D. A. A., et al, 1994. Immune responses in humans and animals to meningococcal transferrin-binding proteins: implications for vaccine design. Infect. Immun. 62:2984-2990; Gotschlich, E. C. 1991. The meningococcal serogroup B vaccine protection trials: concluding remarks at the report meeting second day. NIPH Ann. 14:247-250; Noronha, C. P., et al., 1995. Assessment of the direct effectiveness of BC meningococcal vaccine in Rio de Janerio, Brazil: a case-control study. Int. J. Epidemiol. 24:1050-1057; Boslego, J. W. Et al. 1995. Efficacy, safety, and immunogenicity of a meningococcal group B(15:P1.3) outer membrane protein vaccine in Iquique Chile. Chilean National Committee for Meningococcal Disease. Vaccine. 13:821-829).
Outer membrane complexes as well as individual outer membrane components, including lipids, phospholipids, lipopolysaccharides and proteins, have been evaluated as potential N.m. B vaccines (Dalseg, R., et al., 1995. Group B meningococcal OMV vaccine as a mucosal immunogen. Clin. Immunol. Immunopathol. 76:S93; Hoiby, E. A., et al., 1991. Bacteriocidal antibodies after vaccination with the Norwegian meningococcal serogroup B outer membrane vesicle vaccine: a brief survey. NIPH Ann. 14:147-156; Jarvis, G. A., and J. M. Griffiss. 1991. Human IgA1 blockage of IgG-initiated lysis of N.m. is a function of antigen-binding fragment binding to the polysaccharide capsule. J. Immunol. 147:1962-1967). While outer membrane bleb-based and outer membrane vesicle-based (OMVs) vaccines are able to elicit at least some degree of bactericidal antibodies and mild cross-strain protection in young children, these vaccines are difficult and problematic to prepare which renders them impractical as commercial vaccines.
The class I and class II outer membrane porin proteins (PorA, PorB), the iron-inducible transferrin/lactoferrin-binding proteins, the class V opacity adhesin(s), and the class I/II surface fimbrial adhesins (pili) have been suggested as possible subunit vaccine candidates. Various investigators have shown that although all these proteins are immunogenic and some even elicit bacteriocidal activity, they all show a very high degree of antigenic variability. The surface-exposed strain-variable domains of these proteins also correspond to neutralizing B-cell epitopes (Poolman, J. T. 1995. Surface structure and secreted products of meningococci. In: Meningococcal Disease. Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). Due to the antigenic variation among the major outer membrane proteins of the meningococci, these proteins confer limited cross-strain protection and are thus not suitable as cross-protective subunit vaccines. Thus, an effective cross-protective N.m. type B subunit vaccine candidate must be highly conserved as well as immunogenic.
The HtrA protein has been identified as a virulence factor for several bacterial pathogens including, Yersinia enterocolitica, Brucella abortus, and Salmonella typhimurium. In some but not all organisms HtrA appears to be a stress-responsive protein, possibly contributing to the organisms survival under oxidative challenge and/or at elevated temperatures. The exact role HtrA plays during the pathogenesis process has not yet been fully defined. Bacteria-host cell interaction and the resulting signal transduction events that are triggered in the pathogen may promote expression of the HtrA protein. The E. coli and H. influenzae HtrA proteins, including the Hin47 protein described in U.S. Pat. Nos. 5,679,547 and 5,721,115, both of which are incorporated herein by reference in their entireties, have been shown to be serine proteases and possess three relatively conserved domains that house the catalytic residues H, D and S.
HtrA is a virulence factor, having serine protease activity, which has recently been identified as a target for the development of anti-bacterial agents against gram negative bacterial pathogens. (Jones and Hruby, 1998, New targets for antibiotic development: biogenesis of surface adherence structures, DDT Vol.3(11)495-504; Barrett and Hoch, 1998, Two-component signal transduction as a target for microbial anti-infective therapy, Antimicrobial. Agents and Chemother. 42(7):1529-1536; Fabret and Hoch, 1998, A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy, J. Bacteriol., 180(23):6375-6382).
Citation or identification of any reference in this section or any other section of this application shall not be construed as an indication that such reference is available as prior art to the present invention.
One object of this invention is to identify and provide a novel and highly conserved protein (referred to hereafter and in the claims as xe2x80x9cNMASPxe2x80x9d) from Neisseria meningitidis. The protein of the present invention has a molecular weight of approximately 40-55 kD, and has limited similarity (xcx9c36% identity) BLAST Program (Altschul et al., 1990, J. Molec. Biol. 215:403-10; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) with data entered using FASTA format; expect 10 filter default; description 100, alignment[overall), to the DegP (HtrA) protein of E. coli and has not been previously identified in any Neisseria meningitidis. The protein sequence which is another object of this invention has similarity to several DegP/HtrA-like seine proteases from two other bacteria and these sequence homologies have not been previously reported for any Neisseria meningitidis. 
The invention is based, in part, on the surprising discovery that Neisseria meningitidis, and various strains and cultivars thereof, have a protein, NMASP polypeptide, which is about 40 kD to about 55 kD in molecular weight, preferably about 44 kD to about 53 kD.
The present invention encompasses the NMASP polypeptide of Neisseria meningitidis in isolated or recombinant form. The invention encompasses a purified NMASP polypeptide, polypeptides derived therefrom (NMASP-derived polypeptides), and methods for making said polypeptide and derived polypeptides. The invention also encompasses antisera and antibodies, including cytotoxic or bactericidal antibodies, which bind to and are specific for the NMASP polypeptide, NMASP-derived polypeptides and/or fragments thereof.
The invention further encompasses pharmaceutical compositions including prophylactic or therapeutic compositions and which may be antigenic or immunogenic compositions including vaccines, comprising one or more of said polypeptides, optionally in combination with, fused to or conjugated to another component, including a lipid, phospholipid, a carbohydrate including a lipopolysaccharide or any of the proteins, particularly any Neisseria, Moraxella, Pseudomonas, Streptococcus or Haemophilus protein known to those skilled in the art. The invention further encompasses pharmaceutical compositions including prophylactic or therapeutic compositions, which may be antigenic, preferably immunogenic compositions including vaccines, comprising one or more of the NMASP polypeptide and NMASP-derived polypeptides and an attenuated or inactivated Neisseria, Moraxella, Pseudomonas, Streptococcus or Haemophilus cultivar or an attenuated or inactivated Neisseria cultivar expressing NMASP polypeptide in a greater amount when compared to wild-type Neisseria.
The invention additionally provides methods of inducing an immune response to Neisseria meningitidis in an animal and methods of treating or preventing an infection caused by Neisseria meningitidis in an animal.
The invention further provides isolated nucleotide sequences encoding the NMASP polypeptide, NMASP-derived polypeptides, and fragments thereof, vectors having said sequences, host cells containing said vectors, recombinant polypeptides produced therefrom, and pharmaceutical compositions comprising the nucleotide sequences, vectors, and cells. The nucleotide sequence of the NMASP nucleic acid is shown in SEQ ID NO:1. A deduced amino acid sequence of the open reading frame of NMASP is shown in SEQ ID NO:2.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a NMASP peptide or polypeptide or the DNA sequences of the invention encoding same comprising: contacting the DNA or polypeptide to assess the binding or other interaction, such binding or interaction being associated with a binding or interaction of the DNA or polypeptide with the compound and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the DNA or polypeptide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the DNA or polypeptide. In accordance with another aspect of the invention, there are provided NMASP agonist or antagonists, preferably bacteriostatic bacteriocidal agonists or antagonists.
One advantage of this invention is that antibody generated against the newly discovered NMASP polypeptide of the present invention, in an animal host will exhibit bactericidal and/or opsonic activity against many Neisseriae meningitidis strains and thus confer broad cross-strain protection. Bactericidal and/or opsonic antibody will prevent the bacterium from infecting the host and/or enhance the clearance of the pathogen by the host""s immune system. Neisseria meningitidis antibody bactericidal activity is the principal laboratory test that has been correlated with protection in humans and is the standard assay in the field as being predictive of a vaccine""s efficacy against Neisseria meningitidis infections. Bactericidal antibodies are particularly important for N.m. vaccines because there is no natural animal host other than humans and thus there is no relevant predictive animal model of disease.
Nucleotide or nucleic acid sequences defined herein are represented by one-letter symbols for the bases as follows:
A (adenine)
C (cytosine)
G (guanine)
T (thymine)
U (uracil)
M (A or C)
R(A or G)
W (A or T/U)
S (C or G)
Y (C or T/U)
K (G or T/U)
V (A or C or G; not T/U)
H (A or C or T/U; not G)
D (A or G or T/U; not C)
B (C or G or T/U; not A)
N (A or C or G or T/U) or (unknown)
Peptide and polypeptide sequences defined herein are represented by one-letter or three symbols for amino acid residues as follows:
The present invention may be more fully understood by reference to the following detailed description of the invention, non-limiting examples of specific embodiments of the invention and the appended figures.